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研究生:劉凱文
研究生(外文):Kai-Wun Liou
論文名稱:細菌口引口朵乙醯天門冬胺酸水解酉每經由多精胺酸胜月太運送至植物中之活性研究
論文名稱(外文):Studies of bacterial indole-3-acetyl-L-aspartic acid hydrolase activity in plants through arginine-rich intracellular delivery
指導教授:周志青
指導教授(外文):Jyh-Ching Chou
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:74
外文關鍵詞:AIDIAASPH
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中文摘要
IAA (Indole-3-acetic acid) 是植物之中一種重要的賀爾蒙,它在植物的生長、分化扮演著重要的角色,IAA-Asp (IAA-Aspartic acid) 是首先被發現的IAA 醯胺鍵結共軛物,研究顯示 IAA-Asp 是植物體中 IAA 進行non-decarboxylative oxidation代謝路徑的一個重要中間產物。 Tat 是一個HIV-1的蛋白,Tat 蛋白質是一個具有86個胺基酸的蛋白質,而其中的11個蛋白質更是具有運送蛋白質進入細胞的功能。本實驗中,以 Tat 藉由共價或非共價的鍵結方式將 IAASPH (IAA-Aspartic acid hydrolase) 送入綠豆種子細胞中,藉蛋白質 IAASPH 或 IAASPH-Tat 水解綠豆種子中 IAA-Asp ,造成 IAA 降解路徑受阻,而使 IAA 在綠豆種子中累積,進而影響綠豆種子根生長。最後,以 HPLC 分析綠豆種子中 IAA 含量變化,結果顯示,共價或非共價的運輸皆會造成種子根生長受抑制,而單獨蛋白 Tat、IAASPH 並不會有抑制情形,而種子在0、12、24小時各時間點中,每顆種子所含 IAA 重量皆較正常生長種子為高,表示 IAA 累積現象正在種子中發生,而24、36、48小時各時間點中的IAA 則逐漸減少。
Abstract
IAA (Indole-3-acetic acid) is an essential hormone in plants. It plays an important role in plant growth and development. IAA-Asp (IAA-Asparic acid ) was the first naturally occurring IAA amide conjugate to be chemically characterized. Earlier studies indicated that IAA-Asp is a key intermediate in plant’s IAA non-decarboxylative oxidation pathway. Tat is an HIV-1 protein. The Tat protein contains 86 amino acids, and 11 of them provide the function of delivering protein into cells. This experiment is to deliver the IAASPH (IAA-Asparic acid hydrolase) into the mung bean seed with Tat protein through covalent or noncovalent fashion. Through IAASPH or IAASPH-Tat to hydrolyze IAA-Asp, the IAA degration pathway could be indirectly blocked and therefore accumulate the IAA in the mung bean. Finally, we analyze the IAA level by HPLC. The results indicated that both covalent and noncovalent delivery could inhibit the root growth but the Tat or IAASPH only could not. In 0, 12 and 24 hours, the IAA concentration is higher than the control indicated that IAA accumulation was caused by the IAASPH or IAASPH-Tat. At 24, 36 and 48 hours, IAA level reduced gradually.
目次
中文摘要..................................................Ⅰ
Abstract..................................................Ⅱ
縮寫表....................................................Ⅲ
目錄......................................................Ⅳ
圖、表目錄................................................Ⅶ

壹、前言
一、IAA...................................................1
1. IAA 的合成與代謝...................................... 2
2. IAA 共軛物的形成與水解................................ 2
3. IAA-Asp 與 IAA-Asp 水解酶..............................4
二、AID 胜肽..............................................5
1.蛋白質的共價與非共價運輸方式............................6
貳、目的..................................................7
参、材料與方法............................................8
一、建構質體 DNA..........................................8
二、質體 DNA 的轉形作用..................................10
三、蛋白質大量表現.......................................11
四、蛋白質純化...........................................12
五、蛋白質定量...........................................12
六、蛋白質純度分析.......................................13
七、西方轉印法...........................................14
八、酵素活性分析.........................................15
九、蛋白質的共價與非共價運輸.............................16
1.蛋白質的非共價運輸(Tat, R9 與 GFP)...................16
2.蛋白質的共價運輸(IAASPH-Tat).........................17
3.蛋白質的非共價運輸(IAASPH 與Tat )....................17
十、共軛焦顯微鏡的植物組織觀察...........................18
十一、綠豆種子中 IAA 的萃取、分析........................18
十二、不同濃度 IAA 處理綠豆種子..........................19
肆、結果.................................................20
一、DNA 序列的質體 DNA,pIAASPH-EcoRI、pIAASPH-TAT或pIAASPH-R9.......................................................20
二、獲得正確且可以使用的蛋白質...........................21
三、IAA-Asp 水解酶具有水解IAA-Asp 的活性.................22
四、外加不同濃度 IAA 對綠豆種子生長並無明顯的影響........22
五、蛋白質 Tat 或 R9 可經非共價性運輸 GFP 蛋白進入綠豆根尖細胞.......................................................23
六、蛋白質 Tat以共價性鍵結方式將 IAASPH 運送入綠豆種子中後,會對綠豆種子生長造成顯著影響.............................23
七、蛋白質 Tat以非共價性鍵結方式將 IAASPH 運送入綠豆種子中後,會對綠豆種子生長造成顯著影響.........................24
八、以非共價性運輸 IAASPH 至綠豆種子中,造成綠豆種子在生長初期游離型式 IAA 濃度增加..................................26
伍、討論.................................................29
陸、參考文獻.............................................33
柒、圖、表...............................................40
參考文獻
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